Multi-walled carbon nanotubes wrapped with polyvinylpyrrolidone can control the leaf yellowing of Alstroemeria cut flowers

The rapid yellowing of the leaves on cut flowers with leafy stems severely limits their vase life and commercial value. In this study, the effect of a composite of multi-walled carbon nanotubes (MWCNTs) and polyvinyl pyrrolidone (PVP) on the longevity of cut Alstroemeria flowers (Alstroemeria hybrida) was investigated to obtain a solution to this problem. A range of MWCNTs/PVP composite concentrations (0, 3, 6, and 9 mg L−1) was applied in a vase solution (for 24 h) as pulse treatments. Our findings indicate that the composite of MWCNTs and PVP exhibits excellent dispersibility in a vase solution. The results demonstrate that a 3 mg L−1 MWCNTs/PVP concentration was the most effective, extending the vase life of cut Alstroemeria flowers by up to 27 days. Pulsing with MWCNTs/PVP delayed the onset of floret abscission and leaf yellowing by 5 and 18 days, respectively. Additionally, when MWCNTs/PVP solution was applied to cut stems, water uptake remained consistently greater than that of the control. Additionally, MWCNTs/PVP increased the total chlorophyll content, soluble protein content, and POX enzyme activity of leaves while decreasing the malondialdehyde (MDA) content. The results indicate that this composite exhibited antimicrobial activity against gram-positive and -negative bacteria, particularly at a concentration of 3 mg L−1. This study demonstrated that adding MWCNTs/PVP to a vase solution of Alstroemeria cut flowers increased their longevity with minimal leaf yellowing symptoms compared to untreated cut stems. As a result, this nanocomposite can be used safely and effectively in vase solutions and in combination with other preservatives.

). After 8.33 days, the control florets had already been abscised, whereas cut flowers treated with MWCNTs/PVP (at 3 mg L −1 ) remained intact for an additional 14 days before falling. After 12 days, abscission of the first floret was observed in all of the cut stems and was treated with 6 and 9 mg L −1 MWCNTs/ PVP. Leaf yellowing. Compared to the control, all MWCNTs/PVP pulse treatments delayed the yellowing of cut Alstroemeria (Table 1). MWCNTs/PVP pulse treatment at a concentration of 3 mg L −1 delayed the yellowing of cut Alstroemeria leaves. Additionally, this treatment tripled the control's leaf longevity and preserved the www.nature.com/scientificreports/ leaves green color for 30 days. Before day 12, the entire foliage on control stems appeared severely wilted, and most leaves had yellowed. On day 20, cut stems treated with 6 and 9 mg L −1 MWCNTs/PVP remained green. An intriguing facet of this case of Alstroemeria leaf yellowing was the variation in leaf yellowing patterns between Alstroemeria species. The leaves of nanocomposite-treated plants gradually lost their green color from the bottom of the cut stem upward (and near the florets), whereas all leaves of the cut stem were yellowed when flowers were treated with distilled water (Fig. 2).
Relative fresh weight. Table 2 illustrates the variation in the fresh weight of cut stems during the vase life.
The relative fresh weight decreased gradually except on days 1 and 3, when it increased rapidly, particularly in flowers kept in 3 mg L −1 MWCNTs/PVP. The cut stems in this treatment maintained a higher fresh weight than at the start of the experiment. Cut stems treated with 9 mg L −1 MWCNTs/PVP exhibited a lower fresh weight than stems treated with 6 mg L −1 MWCNTs/PVP. After 5 days, cut stems kept in water (control) began to lose weight and, after 9 days, had a lower fresh weight than their initial value.
Water uptake. In this study, it was observed that water uptake in the vase solution followed a pattern similar to the relative fresh weight of the cut flower, as the water absorption rate initially increased but then gradually    www.nature.com/scientificreports/ Leaf soluble protein content. Table 5 illustrates the variations in soluble protein content of cut flowers. MWCNTs/PVP pulsing affected the protein content. Flowers kept in 3 mg L −1 MWCNTs/PVP consistently exhibited the highest total soluble protein contents in leaves compared to the other treatments. On day 9, the total protein content in water-treated flowers (control) was 6.57 g kg −1 , and soluble protein content decreased by 52% from the initial level. On day 11, the protein content of leaves from cut stems kept in 6 and 9 mg L −1 MWCNTs/PVP decreased by 35% and 43%, respectively, relative to the initial level.
POX enzyme activity. The POX enzyme activity was increased in MWCNTs/PVP treated flowers compared to untreated cut Alstroemeria. Conversely, no change in the activity of the POX enzyme was observed during the vase life of control cut flowers (Table 6). However, it subsequently decreased in water-treated flowers on day 9 to 0.42 mmol min −1 kg −1 .

Malondialdehyde (MDA). MDA accumulation increased exponentially over the vase life of water-treated
cut Alstroemeria, reaching a peak of 17.33 µmol kg −1 on day 9. MDA levels gradually increased in flowers pulsed with MWCNTs/PVP (3 mg L −1 ) and peaked on day 11 (Table 7). Flowers treated with 6 or 9 MWCNTs/PVP reduced MDA accumulation compared to the control; the effect was more pronounced with the 6 mg L −1 concentration than with the 9 mg L −1 concentration.
Stem end bacterial population. The gram-negative bacterial population was increased in the stem ends of untreated cut Alstroemeria flowers; the greatest increase was observed in control flowers (Table 8). Compared to the control, MWCNTs/PVP (3 mg L −1 ) treatment reduced the population of bacteria (Fig. 3). The highest reduction in gram-negative bacteria was observed with 3 mg L −1 MWCNTs/PVP, with 98.4% of bacteria exhibiting a low count (8 × 10 4 CFU mL −1 ). Additionally, 3 mg L −1 MWCNTs/PVP demonstrated the highest reduction percentage in the gram-positive bacterial population at 91%, as the stem end had a bacterial count of 9 × 10 5 CFU mL −1 . Overall, the effect of nanocomposite on gram-negative bacteria inhibition was greater.
Scanning electron microscopy (SEM). SEM analysis revealed that after the evaluation period, MWC-NTs/PVP was detected in the stem of cut Alstroemeria and deposited on the stem's internal surface (Fig. 4). When applied in a vase solution, the images revealed that the MWCNTs/PVP could be allocated to stems and was easily visible within the stem. Therefore, this MWCNTs/PVP composite may serve as an effective scaffold for

Discussion
Carbon nanotubes are well-dispersed in aqueous media and do not aggregate 49 . Given that the MWCNTs are delivered to the plant's stem via water, ensuring they are evenly distributed throughout the vase solution was critical. As previously described in the "Materials and methods" section, this was facilitated by the MWCNTs/PVP    34,50 . However, at a high concentration of MWCNTs/PVP (9 mg L −1 ), several black spots in the cut stem end demonstrated that the MWCNTs aggregated within the xylem, which could potentially have detrimental effects such as inhibiting nutrient and water transport. This method confirms that the effect of MWCNTs/PVP on cut flower longevity was dose-dependent within a narrow dose range. Our findings, similar to those in carnation 51 , indicate that SWCNT has a toxic effect on the vase life of carnation at higher concentrations. Di Zhang et al. 51 explicated that SWCNT cannot be absorbed or transported by plant vascular tissue and can therefore cause vascular tissue blockage.
Moreover, we demonstrated for the first time that MWCNTs/PVP delayed the yellowing of cut Alstroemeria leaves. According to Table 4, the total chlorophyll concentration in the leaves of cut stems treated with the MWCNTs/PVP solution was higher than in the leaves of control plants. Photosynthesis requires the chloroplast structure to be stable 52 . One possible explanation for this observed effect could be that MWCNTs are capable of penetrating the chloroplast, exhibiting the MWCNTs' ability to stabilize the chloroplast structure 53 .
Although little is known about the interaction of CNTs with plants, some mechanisms underlying the effect of CNT on plants are known. In line with our findings, Hu et al. 52 observed that MWCNTs could increase the chlorophyll content and promote Zea mays growth by regulating carbon and nitrogen metabolism. CNTs travel through the symplastic and apoplastic pathways 54 until they reach the mesophyll cells, where they modify the chloroplast absorption profile by stimulating light absorption in the ultraviolet, green, and near-infrared ranges of the spectrum, thereby increasing the chlorophyll content 6 . Additionally, electron transfer between carbon nanotubes and thylakoids has been reported 55 . One mechanism for the beneficial effects of MWCNTs has been their ability to promote the expression of genes involved in photosynthesis, thereby enhancing photosynthetic efficiency 56 . In addition, the increase in total chlorophyll content observed at low doses of MWCNTs/PVP (Table 4) may result from the increased photosynthetic activity 57,58 . Velikova et al. 59 observed that SWCNTs could alter pea plants' leaf micromorphology and chloroplast ultrastructure, thereby affecting their photosynthetic activity.
Cut flower dehydration is a fundamental impediment to preserving their quality. Maintaining a balanced water supply and limiting transpiration and water loss from whole-cut flowers contribute to improving the postharvest quality of flowers 60 . Our findings suggest that MWCNTs can increase water uptake, resulting in increased fresh weight in the cut stem. MWCNTs have previously been shown to penetrate seed coats, promote germination and growth, and improve plant water uptake 22 . Furthermore, it can aid in advancing plant anatomy and physiology 20,61 . CNTs may facilitate water and nutrient uptake by increasing the size of the xylem cell and enhancing the vascular region.
Previous studies suggest that CNTs create new pores for water permeation and enhance capillary action. CNTs stimulate the conduction processes in the stem by forming a network of capillaries across the vascular tissue (xylem and phloem cells) 20 . Also, CNTs are transported within the vascular tissue via transpiration through leaves 62 . According to Lahiani et al. 63 , MWCNTs stimulate the expression of water channel genes (Aquaporins). One possible explanation for the enhancement of water uptake is that CNTs' passive transport is facilitated by their size, charge characteristics (predominantly negative), and interactions with lipids 62,64 . Following wounding www.nature.com/scientificreports/ in cut flower stem, the wound-induced responses activate and deposition of polyphenolic compounds, including lignin (which causes vascular occlusion), at the site of the wound occurred 65,66 . Some studies have shown that the application of PVP in a culture medium cause elimination of phenolic compounds and reduces the browning of explants 67,68 . Because PVP can adsorb polyphenols, it can be argued that PVP, as a polyphenol absorbing agent, is effective in reducing vascular occlusion and has positive synergistic effects with MWCNTs in improving water uptake by the stem.
The MWCNTs used in this study increased the soluble protein content and the peroxidase enzyme activity (Tables 5 and 6). On the other hand, with increasing MWCNTs concentration (6 and 9 mg L −1 ), biochemical activities failed to increase. This shows that the concentration of 3 mg L −1 is optimal for increasing Alstroemeria's postharvest quality. It is evident that nanotubes should be used in a certain range for the desired effect. As a result, excessive exposure to MWCNTs/PVP may be toxic to the plant's biochemical and physiological processes. Exposure to MWCNTs at higher concentrations could damage various metabolic activities, consequently leading to the accelerating senescence of plants. In this study, as expected, the application of MWCNTs/PVP in vase solution led to a low level of MDA in leaves (Table 7), which may result from the material's ability to prevent lipid peroxidation.
Similarly, Ren et al. 69 found that single-wall carbon nanotubes increased antioxidant system activity while lowering MDA levels. Zhao et al. 53 demonstrated that exogenous MWCNTs maintain a low level of reactive oxygen species (ROS) and regulate the expression and activity of enzymes involved in the ASA-GSH cycle 53 , increased antioxidant activity by removing reactive oxygen species, resulting in increased protein stability, decreased lipid peroxidation, increased membrane integrity, and improved plant quality 69,70 .
CNTs can extend the vase life of cut flowers as a result of their high antimicrobial activity. MWCNTs/PVP reduced the bacterial population (particularly gram-negative bacteria) at the cut stem end in this study. Other studies have observed comparable results. Chen et al. 71 demonstrated that MWCNTs exhibit inhibitory activity against various microorganisms. Numerous factors affect the antimicrobial properties of CNTs. Modified CNTs may demonstrate potent antimicrobial and anti-adhesive properties 17 . Other than the type of CNT nanocomposites used, the most important factors influencing the antimicrobial efficacy of CNTs are mechanical damage caused by cell disruption, the release of intracellular content, and the generation of oxidative stress. 17,72 . Our findings corroborated with Fang et al. 73 , who demonstrated that reducing bacterial blockages in the xylem vessels improves the water uptake and vase life of cut flowers. Also, Cai et al. 67 reported that adding PVP to the culture medium of petal explants of Paeonia lactiflora was an effective method of inhibiting contamination. Due to their antimicrobial properties, MWCNTs/PVP composites have great potential for use in postharvest cut flowers.

Conclusion
In conclusion, a solution containing a well-dispersed MWCNTs/PVP composite is a novel method for facilitating plants' uptake of this nanotube. The present findings unequivocally demonstrate that MWCNTs delay leaf yellowing by affecting water absorption, chlorophyll content, and antimicrobial properties, all of which are critical for the longevity of cut flowers. Unlike control plants, where leaf yellowing occurred abruptly on the cut stem, nanocomposite-treated plants gradually lost their green color from their base upward. Our research sheds new light on the interaction between MWCNTs, and Alstroemeria cut flowers and establishes the rationale for MWCNT application postharvest. This study represents a reliable advancement in increased water uptake in cut stems, resulting in beneficial physiological responses because cut flowers retain their ornamental value and quality. Moreover, additional research on MWCNTs' interactions with cut flower systems is necessary to elucidate their longevity and toxicity mechanisms.

Materials and methods
Plant material. The present investigation involving plant materials adheres to all applicable institutional, national, and international guidelines. In addition, the study does not utilize particular plant materials from the experimental area. The plant materials were identified using a catalog available at the greenhouse located in Ashianehsabz, Tehran, Iran. The experimental material consisted of freshly cut, commercial-grade Alstroemeria (Alstroemeria hybrida) 'Sarah' flowers. Fresh flowers were harvested in the morning when the first florets began to open, and others had developed 50% color (commercial stage). Cut stems were immediately transferred to the laboratory of Ilam University and trimmed to a length of 40 cm under deionized water before treatment to avoid stem air blockages. Afterward, lower leaves were stripped from cut stems and placed in vase solutions. Each cut stem was placed individually in the vase containing the vase solution. Finally, all cut stems were kept in a vase life room with a controlled temperature of 20 ± 2 °C, 65 ± 5% relative humidity, and a light intensity of μmol m −2 s −1 under a daily photoperiod of 12 h. www.nature.com/scientificreports/ and then transferred to deionized water in vases. Cut Alstroemeria stems were kept in 250 mL glass vases (Erlenmeyer flask) containing 100 mL of deionized water as vase solutions. The vase solutions were changed daily. The mouths of the vases were covered with aluminum foil paper to prevent contamination and reduce evaporation.

Evaluation of vase life, floret abscission, and leaf yellowing.
The vase life of cut Alstroemeria flowers was determined by the length of time between the first day the flowers were placed in the vase solution and the last day when half of the florets or leaves had wilted, discolored, or abscised 75 . The first abscised floret of individual inflorescences was recorded to determine the timing of floret abscission during vase life. Leaf yellowing was defined as the number of days required to discolor 50% of the leaves on a cut stem 75 .
Measurement of relative fresh weight and water uptake. Throughout the vase life of each replicated cut flower, the daily relative fresh weight (RFW) was determined using an analytical balance and expressed as a percentage of the initial fresh weight (FW) on day 0 76 . Daily changes in water uptake (L kg −1 ) of the cut stems were calculated from volume losses in the vase solutions and expressed as the difference between water uptake and fresh weight change 76 .
Chlorophyll content determination. Chlorophyll content in Alstroemeria leaves was determined through the Lichtenthaler and Buschmann 77 method. Absorbance was measured using a spectrophotometer (Analytik Jena AG, Specord 50 Plus, Germany) at 663 nm and 647 nm. Total chlorophyll content was expressed as g per kg on a fresh weight basis (g kg −1 ).
Determination of leaf soluble protein content, POX enzyme activity, and malondialdehyde (MDA). Initially, 0.2 g of frozen leaf tissue was ground in liquid nitrogen using a mortar and pestle to determine enzyme activity. The POX activity (EC 1.11.1.7) was determined by measuring the increase in absorbance at 470 nm every 15 s for 1 min 78 . The enzyme activity was expressed in mmol within 1 min per kg fresh weight basis (mmol min −1 kg −1 ). The protein concentration was measured using the Bradford 79 method. The absorbance was measured using bovine serum albumin (BSA) as a standard at 595 nm. Total protein content was expressed as g per kg on a fresh weight basis (g kg −1 ). The MDA content was estimated according to Heath and Packer 80 . Afterward, the MDA content was determined via a spectrophotometer at 532 and 600 nm and expressed as μmol per kg on a fresh weight basis (µmol kg −1 ).

Measurement of stem end bacterial population.
After the experiment, the 2-cm-long cut stem end of Alstroemeria was crushed and homogenized, then diluted with water. The extract was then divided into aliquots (0.1 mL) and spread on nutrient agar plates. An incubator programmed to 37 °C was used to maintain the Petri dishes for 48 h. Gram-negative bacteria were cultured on Eosin Methylene Blue (EMB) agar, while gram-positive bacteria were cultured on Azide Blood Agar Base. Following that, log CFU mL −1 counts of gram-positive and gram-negative bacteria were performed 81 .

Scanning electron microscopy (SEM) observation of the stem section. The distribution of MWC-
NTs in the cut stem was performed using FE-SEM (MIRA3, TESCAN). Preparation of the samples for imaging was determined according to the Haberman et al. 82 method with different steps of fixation, saturation, and drying. Briefly, stem tissues were fixed in 0.1 M phosphate buffer (pH 7.2) containing 5% glutaraldehyde for 24 h, followed by five washes in phosphate buffer. The stem tissues were gradually saturated with increasing concentrations of ethanol (25%-100%). Stem tissues were then dried in a critical point dryer device for about 2 h. Statistical analysis. ANOVA was used to evaluate the results statistically using SAS 9.4. The LSD multiple range tests (P ≤ 0.05) were used to determine any statistically significant differences between the treatment means. Notably, the mean comparison between treatments was performed until the end of the vase life of the control treatment. All values were expressed as the means ± standard errors of the means.

Data availability
The datasets analyzed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/